Considerable evidence supports a role for parasympathetic stimulation in protection of the heart from the genesis of iarrhythmias. A major complication of diabetes mellitus is the development of an autonomic neuropathy which is associated with an impaired response of the heart to autonomic stimulation. The increased incidence of sudden death in diabetic patients, which is exacerbated by hypercholesterolemia and other cardiac risk factors, may be due at least in part to the increased likelihood of arrhythmia in the presence of an impaired parasympathetic response. The long-term goal of this application is to determine a new relationship between sterols, lipid lowering, insulin signaling and the response of the heart to parasympathetic stimulation. In a cell culture model for lipid lowering, in which embryonic chick atrial cells are cultured in medium supplement with lipoprotein depleted serum (LPDS), we have previously demonstrated that lipoprotein depletion results in a marked enhancement of the negative chronotropic response to parasympathetic stimulation and an increased expression of the M2 muscarinic receptor, the alpha-subunit of the heterotrimeric G-protein, Galpha(i2); and the inward rectifying K channel protein, GIRK1, which mediate the response of the heart to parasympathetic stimulation. Based on preliminary data which suggest that the expression of all 3 of these genes might be regulated by the binding of a sterol regulatory element binding protein, SREBP, a transcription factor which regulates cholesterol, fatty acid and glucose metabolism, to a putative sterol regulatory element in their upstream promoters, we will test 4 major hypotheses: 1.) that the expression of M2, Galpha(i2) and GIRK1/GIRK4 in embryonic chick atrial cells is coordinately regulated by SREBP at the level of transcription; 2.) that lipoprotein depletion stimulates M2, Galpha(i2) and GIRK1/GIRK4 expression and the parasympathetic response of the heart by a dual effect on SREBP: an increase in the level of SREBP and a Ras dependent phosphorylation of SREBP and; 3.) that insulin stimulates M2, Galpha(i2) and GIRK1/GIRK4 expression and the parasympathetic response of chick atrial cells via a similar dual effect on the level and phosphorylation of SREBP and; 4.) that SREBP expression regulates parasympathetic responsiveness of the mouse heart and muscarinic stimulation of IKAch in atrial myocytes from these hearts. These studies would support the existence of a new relationship between lipid lowering, insulin function and the parasympathetic response of the heart which could have important implications for the genesis and treatment of cardiac arrhythmias.